This invention relates to genetically engineered cell lines and cell transplantation therapy. In particular, it relates to oncogene-transformed cell lines useful for transplantation.
Insulin is synthesized, processed and secreted by pancreatic .beta. cells, the major endocrine cell type in the islets of Langerhans that are distributed throughout the pancreas. Pancreatic .beta. cells secrete insulin in response to an increase in extracellular glucose concentration.
The two major forms of diabetes, insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) both are characterized by an inability to deliver insulin in an amount and with the precise timing that is needed for control of glucose homeostasis. The inadequate insulin delivery is caused by: .beta.-cell destruction by autoimmune mechanisms in IDDM, and .beta.-cell dysfunction closely coupled to insulin resistance in NIDDM. Despite these differences in etiology, a common therapeutic goal for the two disorders is to restore the capacity for glucose-mediated insulin release to its normal level.
Treatment of IDDM requires insulin replacement, either by conventional administration of the hormone or by transplantation of insulin-secreting tissue. Since the latter strategy has thus far relied largely on the use of scarce human pancreas as the insulin source, it has not been feasible for general application. Some investigators have proposed the use of xenografts, e.g., porcine, as a means of overcoming the problem of tissue availability. However, the immune barrier to xenografts is formidable, even using techniques such as encapsulation to help them evade the host immune response.
A number of investigators have developed pancreatic .beta.-cell lines using transgenic mice expressing dominant oncogenes, particularly SV40 T-antigen, under control of the insulin promoter {Newgard, C. B., Diabetes, 43:341-350 (1994) and Hanahan, D., Nature, 315:33-40 (1985)}. Mice expressing T-antigen under the control of the rat insulin gene promoter develop .beta.-cell tumors at 12-20 weeks after birth. Unfortunately, most {see Knaack, et al., Diabetes, 43:1413-1417, (1994)} .beta.-cell lines derived from these animals do not retain normal glucose-responsive insulin production {Tal, M., et al., Mol. Cell. Biol., 12:422-32 (1992)}.
In the absence of spontaneously arising cell lines with the desired properties, cell lines can be created by transfer of dominant oncogenes into primary cells {Chou, J. Y., Mol. Endocrinol., 3:1511-14 (1989)}. Such cell lines have been constructed from brain, liver and bone marrow. In some cases, cell lines created in this way retain differentiated functions or the ability to differentiate in vivo {Snyder, E. Y., et al., Cell, 68:33-51 (1992)}. Unfortunately, in many other cases, loss of differentiated function occurs, decreasing the usefulness of the cell line {Jehn, B., et al., Mol. Cell. Biol., 12:3890-3902 (1992)}.
SV40 T-antigen transforms cells by multiple mechanisms including binding and inactivation of the tumor suppressor proteins p53 and retinoblastoma (Rb) {Andersson, A., et al., Transplantation Reviews, 6:20-38 (1992)}. Although SV40 T-antigen has been shown to be sufficient for transformation of rodent cells, human primary cells are more refractory to transformation {Chang, S. E., Biochem. Biophys. Acta, 823:161-94 (1986)}. The frequency of immortalization of human primary fibroblasts transfected with SV40 T-antigen has been estimated to be 3.times.10.sup.-7 per passage in culture {Shay, J. W., et al., Exp. Cell Res., 184:109-18 (1989)}.
Overexpression of the epidermal growth factor (EGF) receptor is often found in pancreatic cancers, as is overexpression of the EGF homologues c-erbB2 and c-rbB3 {Hall, P. A., et al., Cancer Surveys, 16:135-55 (1993)}. Ras genes are among the ost commonly mutated in human cancer, including pancreatic cancer. Of the ras genes, K-ras mutations are present in 80-90% of pancreatic ductal carcinomas {Hruban, R. H., et al., Am. J. Pathol., 143:545-54 (1993)}. Interestingly, H-ras mutations have not been found in pancreatic cancer {Hruban, R. H., et al., Am. J. Pathol., 143:545-54 (1993) and Smit V. T. H. B. M., et al., Nucl. Acid Res., 16:7773-82 (1988)}. H-ras containing an activating mutation, under the control of the elastase promoter, has been expressed in the exocrine tissue of transgenic mice, with consequent tumor formation {Sandgren, E. P., et al., Proc. Natl. Acad. Sci. USA, 88:93-97 (1991) and Quaife, C. J., et al., Cell, 48:1023-34 (1987)}. However, when activated H-ras was expressed specifically in .beta.-cells using the insulin promoter, destruction of islet cells with diabetes occurred in male mice, but not in females {Efrat, S., et al., Mol. Cell. Biol., 10:1779-83 (1990) and Efrat S., Endocrinol., 128:897-901 (1991)}.
As in many other cancers, p53 is commonly mutated in pancreatic cancers. Although c-myc overexpression has not been studied extensively in primary human tumors, it is a potent transforming gene when expressed in the pancreas of transgenic mice.
Gene Transfer Into Primary Cells
A problem with the development of immortalized cell lines from primary cells, and particularly human primary cells, is that these cells are resistant to most methods of gene transfer. Gene transfer into islet cells has been accomplished by electroporation {German, M. S., et al., J. Biol. Chem., 265:22063-22066 (1990)}. However, gene expression was only studied on a transient basis and required dissociating the islets into a single cell suspension. Such treatment is deleterious to the survival of cells from the human pancreas {Beattie, G., et al., J. Clin. Endocr. Metab., 78:1232-40 (1994)}. Adenovirus vectors efficiently infect pancreatic cells {Newgard, C. B., Diabetes, 43:341-50 (1994)}, but maintaining long term gene expression from these vectors has been a problem {Smith, T. A. G., et al., Nature Genet., 5:397-402 (1993)}. Alternatively, transgenic technology may be used. This usually involves expressing an oncogene, usually SV40 T-antigen, under control of the insulin promoter in transgenic animals, thereby generating cell tumors that can be used for propagating insulinoma cell lines {Efrat, S., et al., Proc. Natl. Acad. Sci. USA, 85:9037-41 (1988); Miyazaki, J. I., et al., Endocrinology, 127:127-32 (1990)}. Cell lines derived by transgenic expression of T-antigen in .beta.-cells exhibit variable phenotypes. Some have little glucose-stimulated insulin release or exhibit maximal responses at subphysiological glucose concentrations, while others respond to glucose concentrations over the physiological range. However, the near normal responsiveness of the latter cell lines is not permanent, as continuous cell culture results in a shift in glucose dose response such that the cells secrete insulin at subphysiological glucose concentrations. A detailed discussion of these cell lines is found in Newgard, C. B., Diabetes, 43:341-350 (1994). A human insulinoma cell line has been obtained but it is difficult to maintain in culture and does not produce insulin {Gueli, N., et al., Exp. Clin. Cancer Res., 6(4):281-285 (1987)}.
Retroviral-mediated gene transfer (i.e., the use of retroviruses to deliver genes into cells) is an alternative gene transfer technology which has met with limited success. In this technique, a desired gene is inserted into a retroviral vector to obtain a recombinant virus which is then used to infect target cells. Retroviruses are ribonucleic acid (RNA) viruses. In retroviral-mediated gene transfer, the viral RNA is first converted to deoxyribonucleic acid (DNA) after an RNA virus penetrates a target cell. If the target cell penetrated is a replicating cell (i.e., mitotically active), the DNA will enter the nucleus and integrate into the genome of the target cell. In this integrated form, the viral genes are expressed. Integration of the viral genome into the target cell's genome is an essential part of its replication. Retroviral vectors are extremely efficient at infecting a wide variety of cell types, including primary cells from many tissues {McLachlin, J. R., et al., Prog. Nuc. Acid Res. Mol. Biol., 38:91-135 (1990)}. The major drawback of retroviral vectors is that mitotically active cells are required in order for the retroviral preintegration complex to enter the nucleus and integrate into the genome.
U.S. Pat. No. 5,256,553 to Overell discloses a retroviral vector containing three inserted genes (two oncogenes and at least one heterologous gene) each of which is independently transcribed in an infected cell under the control of its respective transcriptional control sequence. In its Example 1, the patent discloses primary rat embryo fibroblasts (REFs), Balb/3T3, and .psi.2 (.psi.2 is a retroviral packaging cell line derived from 3T3 cells) transformed by two triple promoter retroviral vectors each containing a v-Ha-ras oncogene, a v-myc oncogene, and a neomycin phosphotransferase (neo) gene which confers resistance to G418 antibiotic resistance. Example 2 of the patent discloses two other triple-promoter vectors, similar to those of Example 1 except that instead of the neo gene, these vectors contained hygro (hph) gene which conferred resistance to hygromycin B. The Example 2 vectors were used to transform Balb/3T3 and .psi.2 cells. In Example 3 of the patent, the vectors of Examples 1 and 2 were transfected into .psi.2 cells. Viruses harvested from the virus-producing clones were incubated with Balb/3T3 cells and found to be capable of infecting the cells. However, it must be noted that cellular transformation is a multistep genetic process in all species, but the process differs between human and rodents in the relative refractoriness of human cells to transformation. The reason for this difference is not known. Additionally, primary human cells are often relatively refractory to many methods of stable gene transfer. Together, these facts make the development of human cell lines in vitro difficult. Thus, most human cell lines have been derived from primary cancers that have been adapted to culture in vitro.